Directed assembly of three-dimensional structures with micron-scale features

ABSTRACT

Polyelectrolyte inks comprising a solvent, a cationic polyelectrolyte dissolved in the solvent, and an anionic polyelectrolyte dissolved in the solvent, are described. The concentration of at least one of the polyelectrolytes in the solvent is in a semidilute regime.

RELATED APPLICATIONS

The present patent document is a divisional of U.S. patent applicationSer. No. 11/560,610, which was filed on Nov. 16, 2006, which is adivisional of U.S. patent application Ser. No. 10/463,834, which wasfiled on Jun. 17, 2003. The preceding patent documents are herebyincorporated by reference in their entirety.

BACKGROUND

Three-dimensional structures with micron-scale features have manypotential applications, for example as photonic band gap materials,tissue engineering scaffolds, biosensors, and drug delivery systems.Consequently, several assembly techniques for fabricating complexthree-dimensional structures with features smaller than 100 microns havebeen developed, such as microfabrication, holographic lithography,two-photon polymerization and colloidal self assembly. However, allthese techniques have limitations that reduce their utility.

Two-photon polymerization is capable of creating three-dimensionalstructures with sub-micron features, but from precursors that are notbiocompatible. Many techniques have been developed to fabricatethree-dimensional photonic crystals, but they rely on expensive,complicated equipment or time-consuming procedures. Colloidalself-assembly has also been utilized to make three-dimensional periodicstructures, but controlling the formation of defects is difficult.

One fabrication technique relies on the deposition of viscoelasticcolloidal inks, usually by a robotic apparatus. These inks flow througha deposition nozzle because the applied pressure shears theinterparticle bonds, inducing a breakdown in the elastic modulus. Themodulus recovers immediately after leaving the nozzle, and the inksolidifies to maintain its shape and span unsupported regions. Theparticles in the ink have a mean diameter of about 1 micron, meaningthat it would be impossible for the ink to flow through a 1 microndiameter deposition nozzle without clogging or jamming. In practice,nanoparticle inks (mean diameter˜60 nm) also tend to jam nozzles smallerthan 30 microns, limiting the applicability of viscoelastic colloidalinks to this length scale.

Polymeric solutions are used in nature to fabricate thin filaments.Spiders, for example, derive their silk fibers from a concentratedprotein biopolymer solution that solidifies as it is drawn to form anextremely strong filament. The extensional flow of the solution alignsliquid crystal sheets in the polymer, and the solution gels by addingions as it leaves the spinneret. This process was artificially recreatedby the deposition of the recombinant spider silk biopolymer into a polar“deposition bath” to produce filament fibers with comparable properties.

SUMMARY

In a first aspect, the disclosure provides polyelectrolyte inkscomprising a solvent, a cationic polyelectrolyte, dissolved in thesolvent, and an anionic polyelectrolyte, dissolved in the solvent. Theconcentration of at least one of the polyelectrolytes in the solvent isin a semidilute regime.

In a second aspect, the disclosure provides a solid filament comprisinga complex of a cationic polyelectrolyte and an anionic polyelectrolyte.The filament has a diameter of at most 10 microns.

In a third aspect, the disclosure provides a method of making apolyelectrolyte ink comprising mixing together ingredients that comprisea solvent, a cationic polyelectrolyte, and an anionic polyelectrolyte.The concentration of at least one of the polyelectrolytes in the solventis in a semidilute regime.

In a fourth aspect, the disclosure provides a method for fabricating afilament, comprising flowing the polyelectrolyte ink through a nozzle,and contacting the ink with a deposition bath. The polyelectrolyte inkgels in the deposition bath.

In a fifth aspect, the disclosure provides a method of formingthree-dimensional structure, comprising fabricating a plurality offilaments, each filament fabricated by the method set forth in thefourth aspect.

DESCRIPTION OF THE DRAWINGS

FIG. 1 shows viscosity and elastic modulus of polyelectrolyte mixturesas a function of the mixing ratio of ionizable groups at a constantpolymer volume fraction (Φ_(poly)=0.4).

FIG. 2 shows the elastic modulus of the ink reacted in a water/IPAdeposition bath as a function of IPA concentration in the depositionreservoir.

FIGS. 3A, 3B and 3C are electron micrographs of structures fabricatedthrough the directed assembly of polyelectrolyte inks. (A) Four-layermicrostructure with a missing rod that may be utilized as a waveguide ina photonic crystal. (B) Eight-layer structure with walls showing theink's ability to form spanning and space-filling elements. (C) Radialstructure showing the inks ability to turn sharp and broad angles.

DETAILED DESCRIPTION

The present disclosure provides a method of microstructure fabricationvia deposition of inks that flow through a deposition nozzle of 10micron or less, without clogging or jamming. When deposited in adeposition bath the inks solidify after leaving the nozzle. Theresulting microstructures have features in the micron scale and areamenable to fabrication with biocompatible materials, and are relativelyeasy and inexpensive to make.

The present disclosure includes the three-dimensional fabrication ofstructures with micron-scale features by making use of an ink. Anapplied pressure forces the ink through a deposition nozzle that isattached to a moving x-y-z micropositioner, into a deposition bath thatgels the ink in situ as the micropositioner moves to form atwo-dimensional pattern on the substrate. The nozzle then incrementallyrises in the z (vertical) direction for the next layer of the pattern.This process is repeated until the desired three-dimensional structurehas been created. With this technique, any three-dimensional structurecan be defined and fabricated.

The inks of the present disclosure are concentrated mixtures ofoppositely charged polyelectrolytes, also referred to as polyelectrolytecomplexes (PEC). The PEC contains two oppositely chargedpolyelectrolytes (e.g. poly(acrylic acid) and poly(ethylenimine)). Onepolyelectrolyte is preferably larger than the other, and theconcentration of the larger polyelectrolyte is preferably within thesemidilute regime: the concentration is above the concentration c* thatseparates the dilute from the semidilute concentration regime. Below c*,in the dilute regime, the mixture of polyelectrolytes forms particlesrather than the single phase fluid needed for the deposition of acontinuous filament. Above c*, in the semidilute regime, polymer coilsstrongly overlap with each other, and the mixture of electrolytes may beused for structure deposition.

The ink viscosity is preferably in the range that allows consistent,controllable flow at a modest applied pressure. Preferred viscosityvalues vary between at least 0.05 Pa*sec to at most 600 Pa*sec. Morepreferred viscosity values are at least 0.1 Pa*sec to at most 150Pa*sec. Yet more preferred viscosity values are at least 1 Pa*sec to atmost 20 Pa*sec. Moreover, the ink undergoes a rapid solidificationreaction when it comes in contact with the deposition bath that allowsthe extruded filament to maintain its shape while spanning unsupportedregions of the structure.

Examples of polyelectrolytes that may be used in PEC are poly(acrylicacid), poly(ethylenimine), poly(styrene sulfonate) poly(allylamine)hydrochloride, poly(diallyldimethyl ammonium chloride), poly(4-vinylpyridine), and cationic or anionic surfactants. Electrically oroptically active classes of polymers, for example polyacetylene,polyaniline, polypyrrole, polythiophene, poly(3,4ethylenedioxythiophene) (PEDOT), NAFION® (Du Pont, Wilmington, Del.),polyphenylene vinylene, polyphenylbenzenamine, sulfonatedpoly-p-phenylene azobenzene dye and other organic dyes may be used, andare well suited to applications involving organic LEDs and circuits. Theparent polymers of some of these classes of polymers do not containcharged groups, however, copolymers and derivatives of these classes do;for example charged groups may be introduced through monomers containingsubstituents (which may be protected until after synthesis of thepolymer), or by derivatizing reactive groups (such as hydroxyl groups,or electrophilic addition on phenyl rings).

For biochemical, molecular biological and biomedical applications, suchas biocatalysis, gene manipulation and tissue engineering, biologicalelectrolytes may be used. Example biological polyelectrolytes arepolynucleotides, such as DNA and RNA, peptides, proteins, peptidenucleic acids, enzymes, polysaccharides such as starch and cellulose,acidic polysaccharides such as hemicelluloses (for examplearabinoglucuronoxylan), basic polysaccharides such as poly-(1,4)N-acetyl-D-glucosamine (chitosan), galactans such as agarose,polyuronides such as alginic acid, carrageenans, hyaluronic acid,collagen, fibrin, proteoglycans, polylactic acid, polyglycolic acid,copolymers of organic acids, cationic lipids. Biologicalpolyelectrolytes with both positive and negative charges, for instancezwitterions such as polycarboxybetaine, may also be included in the inkcompositions.

Bioactive molecules may also be incorporated in the ink, for examplecharged or neutral nutrient molecules, molecular messengers such asgrowth stimulants, and cellular adhesion molecules. Molecular probes forbiomolecules such as cellular lipids or cellular membrane proteins,cellular components such as ion channels and receptors, or organellessuch as mitochondria or lysosomes may also be added.

Smaller organic and inorganic species can also be incorporated into theinks, to amounts that do not deleteriously affect the rheologicalproperties of the ink. Examples include nanoparticles, quantum dots,charge neutral polymers, organometallic precursors and biomolecules.These species may interact with the polyelectrolytes to aid in thegelation or remain inert in the ink, depending on their ionic nature.Also, many other polymers may be made into polyelectrolytes throughfunctionalizing the polymer backbone with charged moieties, for exampleamino groups, sulfonate groups, and carboxylic groups.

The molecular weight of the larger polyelectrolyte is preferably highenough to facilitate chain overlap (preferably at least 5000 daltons)but also low enough to form a concentrated ink with a viscosity thatenables flow at moderate pressures (preferably at most 100,000 daltons).The concentration of the ink is preferably high to avoid deformation ofthe structures upon drying. A typical polymer concentration ranges fromat least 5% to at most 95% by weight. More preferably, the concentrationvaries from at least 25% to at most 75% by weight. Yet more preferably,the concentration varies from at least 35% to at most 45% by weight.Most preferable concentrations range from at least 38% to at most 42% byweight.

The larger polyelectrolyte and the smaller polyelectrolyte arepreferably mixed together in a ratio such that one of the charge groupsis in excess (usually the charge group of the larger polymer), yieldinga mixture away from a stoichiometric (1:1) cationic:anionic group ratio.In the vicinity of this ratio, the strong interactions betweencomplementary polyelectrolytes may lead to the formation of kineticallystable, inhomogeneous aggregates, and the complex may form two phases, apolymer-rich aggregate and a polymer-poor fluid.

Once the polyelectrolytes and solvent have been chosen, a phase diagramcan be developed relating the ratio of cationic to anionic groups as afunction of overall polyelectrolyte concentration (in the chosensolvent), with the goal of determining the range for homogeneous inks.This range will be above the dilute/semidilute transition of the largerpolymer and away from a stoichiometric (1:1) cationic:anionic groupratio. The viscosity of the homogeneous inks increases as the polymerconcentration increases and as the mixing ratio approaches 1:1. Theviscosity may thus be controlled for the deposition of inks through avariety of nozzle sizes.

A deposition bath is selected to fabricate three-dimensional structuresthrough a rapid solidification reaction. In these polyelectrolyte inks,the reaction will occur by increasing the strength of the attractionsbetween the oppositely charged polyelectrolytes. This can be achieved,for example, through pH changes, ionic strength changes, solventcomposition changes, or combinations of more than one change. Thereaction produces a filament that is strong enough to maintain its shapewhile spanning unsupported regions in the structure, but also softenough to allow the filament to adhere to the substrate and flow throughthe nozzle consistently.

Deposition baths that induce gelation through pH changes are generallyused when the polyelectrolytes contain acidic and/or basic chargedgroups. The pH change eliminates the excess of one of the charge groups,for instance by ionizing acidic groups that are neutral at the pH of theink. This yields a mixture with a stoichiometric (1:1) cationic:anionicgroup ratio that gels into a filament.

In addition, the pH of the deposition bath may be selected in order toinduce partial dissolution of the deposited filament, while the shape ismaintained. The pH of the bath lowers the bond strength between theoppositely charged polyelectrolytes, leading to the dissolution. Thestructures have a residual charge on the surface, and may be used forthe adsorption of charged nanoparticles.

Coagulation may also be achieved through changes in solvent composition.For example, an aqueous ink may be deposited in a deposition bathcontaining a relatively apolar solvent such as an alcohol. The resultingdrop in dielectric constant leads to an increase in the coulombicattractions between the polyelectrolytes. Also, an apolar solvent thatis a poor solvent for the polyelectrolytes may be chosen, leading toincreased polyelectrolyte/polyelectrolyte bonding. The reaction yields apolyelectrolyte complex precipitate with a positive:negative chargeratio closer to 1:1 than in the unreacted ink, but not to the extent ofthe pH induced reaction. The structures have a residual charge on thesurface, and may be used for the adsorption of oppositely chargednanoparticles.

Moreover, the mechanical properties of the deposited ink are dependenton the composition of the deposition bath. As illustrated in FIG. 2,different percentages of apolar solvents generally yield filaments ofvarying stiffness.

An apparatus for depositing the ink may be manufactured by connecting adeposition nozzle with a diameter of preferably at least 0.1 microns toat most 10 microns to a micropositioner, for example a computercontrolled piezoelectric micropositioner, and an ink reservoir. Thesemicropositioners are used in a variety of devices, such as scanningtunneling microscopes, and are commercially available. Pressure pushesthe ink through the nozzle, or two or more nozzles, and themicropositioner controls the deposition pattern of the filament.Alternatively, the nozzle (or nozzles) may be static, while the stageholding the substrate on which the microstructure is formed may becontrolled by the micropositioner. In another configuration, both thenozzle and the stage may each be controlled by its own micropositioner.Multiple substrates are also possible when multiple nozzles are present.The assembly of structures is then preferably performed using patternscreated in a computer aided design computer program coupled to themicropositioner.

There are many applications for solid PEC structures fabricated withthese materials and methods. The structure may be infiltrated with ahigh refractive index material and subsequently the PEC structureredissolved to form photonic crystals. The ability of the ink to spandistances renders it possible to engineer defects (for example cavitiesor waveguides) into the structure for functional photonic band gapmaterials. The highly porous structures could be used for membranes thatselectively allow small molecules to flow through at a faster rate.Also, screens may be prepared that do not allow cells, or cells largerthan a certain size, to flow through. Screens of this type may be used,for instance, to separate smaller cells from larger cells in a bloodsample. They may also be used for drug delivery systems where a range ofporosities is necessary for controlled release.

The charged complexes may be used as tissue engineering scaffolds forcell adhesion and growth. For instance, copolymers of poly(L-lacticacid) and poly(L-glycolic acid), both anionic polyelectrolytes that havebeen FDA approved as biodegradable polymers, may be combined with one ormore cationic polymers, such as chitosan, to form a biocompatible inkfor tissue engineering applications. To promote cell growth throughoutthe structure, proteins and sugars may be added to the ink to bereleased as the polyelectrolytes dissolve.

Biologically interesting molecules may also be attached to themicrostructures. Examples of these molecules include nucleic acids,polypeptides and other organic molecules. Nucleic acids includepolynucleotides (having at least two nucleic acids), deoxyribonucleicacid (DNA), such as expressed sequence tags (ESTs), gene fragments orcomplementary DNA (cDNA), or intron sequences that may affect genetranscription, such as promoters, enhancers or structural elements.However, the nucleic acid need not be related to a gene or geneexpression, as aptamers (small nucleic acid sequences that specificallybind to a target molecule) can also be used. Ribonucleic acids (RNA) mayalso be used, such as messenger RNA (mRNA), transfer RNA (tRNA) orribosomal RNA (rRNA). Nucleic acids may also be modified; for example,such as substituting a nucleic acid with a non-naturally occurring one,such as inosine. Chemical modifications of nucleic acids, such as thosethat may confer stability or facilitated immobilization upon asubstrate, may also be used. Another example of a modified nucleic acidis a peptide nucleic acid (PNA), which is a nucleic acid mimic (e.g.,DNA mimic) in that the deoxyribose phosphate backbone is replaced by apseudopeptide backbone and only the four natural nucleobases areretained. The neutral backbone of PNAs allows for specific hybridizationto DNA and RNA under conditions of low ionic strength. The synthesis ofPNA oligomers can be performed using standard solid phase peptidesynthesis protocols. Nucleic acids attached to the microstructures maybe used, for example, in diagnostic and prognostic assays, geneexpression arrays, pharmacogenomic assays, etc.

Polynucleotides may be linked to the microstructures throughthiol-mediated self-assembly attachment to gold nanoparticlesincorporated into the microstructures. The gold may be incorporated intothe microstructures either by addition to the undeposited ink mixture orby attachment to the microstructures after deposition, for example viasulfhydril groups present in the ink.

Polypeptides, having at least two amino acid residues, may find use onor within the substrates of the application. Examples of classes ofpolypeptides include antibodies and derivatives, protein hormones (e.g.,human growth hormone and insulin), extracellular matrix molecules, suchas laminin, collagen or entactin; polypeptides involved in signaling,such as phosphatases and kinases; receptors, such as dopamine receptorsand hormone receptors (advantageously may be attached in the nativeformat, or in the case of homodimers, trimers, etc., mixed with otherpolypeptide chains or as single chains), etc. Attaching polypeptides tothe substrates of the disclosure allow for a wide variety ofapplications, including drug screening, diagnostic and prognosticassays, assays that resemble enzyme-linked immunosorbent assays(ELISAs), proteomic assays and even cellular adhesion studies.

Organic molecules also find use on the microstructures. For example,steroid hormones, such as estrogen and testosterone may be attached.Such couplings facilitate for screens for molecules that bind thesemolecules, such as antibodies or aptamers. Likewise, candidate smallmolecule antagonists or agonists may be attached to facilitatepharmaceutical screening.

Entities such as prions, viruses, bacteria, and eukaryotic cells mayalso be attached. Prions are misfolded protein aggregates that canpropagate their misfolded state onto native proteins; examples includethose aggregates that cause mad cow disease (bovine spongiformencephalopathy (BSE)) or Creutzfeldt-Jacob disease. Examples of virusesinclude herpes simplex, orthopoxviruses (smallpox) or humanimmunodeficiency virus. Bacteria that are of interest may include Vibriocholera, Clostridium perfringens, or Bacillus anthracis (anthrax).Eukaryotic cells, such as those isolated as primary cultures fromsubjects or plants, or from cell lines (e.g., those available from theAmerican Type Culture Collection (ATCC); Manassus, Va.), may beimmobilized onto the microstructures for a variety of purposes,including screens for pharmaceuticals, investigations intocell-substrate adhesion, or for the binding of various molecules.

Any ink that gels through a solvent change may be used to assemblethree-dimensional structures from electrically, optically orbiologically active polymers. Inorganic structures may also befabricated by using sol-gel precursors to produce, for example, sensorsor template-free photonic band gap materials.

EXAMPLES 1) Ink Mixtures

A linear polyacid, poly(acrylic acid) (MW˜10,000) and a highly branchedpolybase, poly(ethylenimine) were combined in an aqueous solvent,yielding solutions with a polymer fraction Φ_(poly)=0.4. When thesepolyions were combined, the carboxylate groups of poly(acrylic acid)(PAA) form ionic bonds to the amine groups of poly(ethylenimine) (PEI).The polymers were initially mixed under mildly acidic conditions(pH˜3.6), where the partial charge on the PAA let only a fraction of thepotentially ionizable groups participate in complexation. The Φ_(poly)was maintained constant, and different PAA to PEI ratios yieldedmixtures with varying rheological properties, as illustrated in thephase diagram of FIG. 1. In this figure, the values on the left and theright y-axes indicate values for pure PAA and pure PEI at Φ_(poly)=0.4The dilute-semidilute crossover concentration c* for PAA is indicated onthe bottom x-axis. The two-phase region consists of a dense,polymer-rich phase, and a fluid-like, polymer-poor phase, and data couldnot be obtained in this regime. As the ratio approaches the two-phaseregion, the elastic modulus and the viscosity of the mixtures increases.

A homogeneous, single phase was observed at mixing ratios in the PAA andPEI rich regions. The charge imbalance forms a non-stoichiometric,hydrophilic complex. The two-phase region, near stoichiometric mixingratios, comprises a dense, polymer-rich phase with a stoichiometric,hydrophobic complex and a fluid-like, polymer-poor phase.

The viscosity differences observed at different mixing ratios may beutilized to assemble structures at different length scales. At smallnozzle sizes, a lower viscosity ink may be deposited at modest appliedpressures, whereas larger nozzle sizes generally require more viscousinks in order to obtain flows with controlled rates.

2) Ink Deposition Apparatus

Inks prepared as described in example 1 were loaded in a depositionapparatus for microstructure fabrication. The apparatus comprised aNanoCube™ XYZ NanoPositioning System (Polytec PI, Auburn, Mass.)controlling μ-Tip (World Precision Instruments, Sarasota, Fla.)deposition nozzles, and the ink was dispensed from the apparatus by aModel 800 ULTRA Dispensing System with a 3 ml ULTRA Barrel Reservoirs(EFD, Providence, R.I.).

3) Fabrication of Structures in an Isopropanol and Water

An ink prepared according to the procedure of example 1, with aΦ_(poly)=0.4, a PAA:PEI ratio of 5.7:1, was deposited, at a velocity of20 microns/second and through a 1 micron nozzle, in a deposition bathcontaining a mixture of isopropanol (IPA) and water. The gelation occursdue to a decrease in solvent quality for the polyelectrolytes and anincrease in the coulombic attractions between the ionizable groups,yielding a reacted ink filament. NMR spectroscopic data (not shown)showed no discernable difference in the types of bonds in the reactedand unreacted complexes, indicating that the reaction only causes achange in the number and strength of the bonds. The mechanicalproperties of the reacted ink were highly dependent on the depositionbath, as illustrated in FIG. 2.

4) Fabrication of Structures in a Water Deposition Bath

The experiment of Example 3 was repeated, this time using an ink with aPAA:PEI ratio of ˜4.8 and a deposition bath of deionized water. The pHchange eliminated the excess of the groups bearing a positive charge byionizing acidic groups that were neutral at the pH of the ink. Thisyielded a mixture with a nearly stoichiometric cationic:anionic groupratio that gelled into a filament (Table 1).

TABLE 1 (−:+) charge Polyelectrolyte Carbon Hydrogen Nitrogen ratio PAA39.48  4.80 NA PEI 52.04 11.78 31.68 NA Unreacted PEC 40.12  5.21  2.274.8:1 Reacted PEC 41.92  5.58  2.90 1.1:1

4) Microstructures

FIGS. 3A to 3C show structures fabricated with a 1 micrometer nozzle inan 83% IPA (balance water) deposition bath. FIG. 3A shows an FCTstructure with a missing filament in the middle that could be used as awaveguide in a photonic crystal. FIG. 3B shows an 8-layer structure withwalls, showing the ability to form solid structures as well as spanningelements. FIG. 3C shows a radial structure with porosity at multiplelength scales. The reacted complex is capable of spanning lengths muchgreater than the filament diameter. Waveguides and radial structureswere fabricated, showing the ability to fabricate structural featureswith tight or broad angles. Periodic structures with different featuresizes were also fabricated, wherein the feature size of a structure isthe diameter of its thinnest filament, obtained with different nozzlesand inks (Φ_(poly)=0.4 and PAA:PEI ratio˜5.7:1; Φ_(poly)=0.4 and PAA:PEIratio˜5:1; Φ_(poly)=0.43 and PAA:PEI ratio˜2:1).

If the deposition occurs in a slightly acidic reservoir, partialdissolution of the complex occurs, while the shape is maintained,leading to highly porous structures with lower elasticity. Thestructures have a residual negative charge on the surface, and may beused for the adsorption of nanoparticles.

The microstructures may also undergo thermal treatment and maintaintheir integrity. For example, the microstructures were heated at 5°C./min to 240° C. in air. The temperature was held at 240° C. for 30minutes, and then cooled at 5° C./min. The structures maintained theiroriginal shape and became harder than prior to the heating. Thishardening may be due also to heat-induced inter-polyelectrolyte bondformation, for example amide bonds formed between the carboxyl groups ofthe PAA and the amine groups in the PEI.

1. An apparatus for the fabrication of microstructures, the apparatuscomprising: (1) a nozzle comprising an opening having a diameter of atmost 10 microns; (2) a stage below the nozzle for supporting asubstrate; (3) a micropositioner operably connected to at least one ofthe nozzle and the stage; and (4) an ink reservoir in fluidcommunication with the nozzle.
 2. The apparatus of claim 1, furthercomprising a pressure dispensing system operably connected to the inkreservoir.
 3. The apparatus of claim 1, wherein the micropositioner is acomputer-controlled micropositioner.
 4. The apparatus of claim 1,wherein the opening of the nozzle has a diameter of at most 1 micron. 5.The apparatus of claim 1, wherein the opening of the nozzle has adiameter of at least 0.1 micron.
 6. The apparatus of claim 1, furthercomprising a plurality of nozzles.
 7. The apparatus of claim 6, furthercomprising a plurality of substrates on the stage.
 8. The apparatus ofclaim 1, further comprising a substrate on the stage, the substratecomprising a deposition bath.
 9. The apparatus of claim 8, wherein thedeposition bath is configured to induce gelation of an ink flowedthrough the nozzle.
 10. The apparatus of claim 8, wherein the depositionbath comprises an aqueous solvent.
 11. The apparatus of claim 8, whereinthe deposition bath comprises an apolar solvent.
 12. The apparatus ofclaim 11, wherein the apolar solvent comprises an alcohol.
 13. Theapparatus of claim 12, wherein the deposition bath comprises a mixtureof isopropyl alcohol and water.